Project Summary PAR-18-206: Injectable Hydrogels to Protect Transplanted Cells from Hypoxia Cell transplantation by direct local injection is a promising strategy for many regenerative medicine therapies; however, regardless of clinical indication, the therapeutic potential of this strategy has been drastically limited by inefficient cell delivery and poor long-term survival of transplanted cells. We have recently designed an injectable hydrogel that improves cell delivery by providing (1) mechanical shielding during the injection process to prevent cell membrane rupture, (2) rapid gelation in vivo to localize cells at the intended delivery site, and (3) cell-adhesive ligands that promote the spreading and migration of transplanted cells into the host tissue. In a preclinical model of spinal cord injury (SCI), use of this hydrogel to transplant Schwann cells (SCs) resulted in a significant increase in successful cell delivery, which correlated with improved therapeutic outcomes. However, poor long-term survival of transplanted cells continues to be an unmet challenge due to the hypoxic host environment. Therefore, we propose the development of two orthogonal biomaterial design strategies (a biomechanical strategy in Aim 1 and a biochemical strategy in Aim 2) to create injectable hydrogels that improve transplanted cell delivery and promote long-term survival in hypoxia. These materials, named SHIELD (Shear-thinning Hydrogels for Injectable Encapsulation and Long-term Delivery) are fully chemically defined to facilitate future FDA studies. As a proof of concept, SHIELD will be evaluated in a preclinical model of SCI, where transplanted SC therapies are known to suffer from significant hypoxic cell death. In Aim 1, we evaluate the hypothesis that matrix mechanics can alter the pro-survival secretome of encapsulated cells, thereby creating soluble, autocrine signals that improve hypoxic survival. Cells will be encapsulated in SHIELD materials with a range of stiffness, cultured under normoxic and hypoxic conditions (5% and 1% O2, respectively), and assessed for viability, proliferation, secretion of neurotrophins and growth factors, and markers of cell necrosis (cyclophilin A and fodrin breakdown product) and apoptosis (caspase-3 and TUNEL). As a parallel approach, in Aim 2, we evaluate the hypothesis that sustained, localized delivery of pro-survival factors can be achieved through the design of stabilized, lipid-vesicle depots that physically crosslink into our injectable hydrogel. The multi-lamellar lipid capsules are stabilized by inter-bilayer covalent crosslinking, and the degree of crosslinking is used to tune the release rate. Thus, this modular design strategy can be used to independently control the delivery kinetics of multiple pro-survival factors. Encapsulated cells will be evaluated as in Aim 1. In Aim 3, we validate our in vitro findings in a preclinical rat model of cervical, contusive SCI with SC transplantation. SC survival and distribution, native tissue response, neuro-regeneration, and functional forelimb recovery will be assessed. In summary, because the success of cell-based regenerative medicine therapies hinges on the survival of transplanted cells, technologies that directly address cell death by hypoxia can significantly improve clinical outcomes.